A large national outbreak of chikungunya virus (CHIKV) was recently reported in Thailand. While dengue virus (DENV) infection tends to occur year-round with an upsurge in the rainy season, Zika virus (ZIKV) also circulates in the country. The overlap in the distribution of these viruses increased the probability of co-infections during the heightened CHIKV activity. By examining 1806 patient serum samples submitted for CHIKV diagnostics from October 2018-February 2020 (511 CHIKV-negatives and 1295 CHIKV-positives), we used real-time reverse transcription-polymerase chain reaction to identify DENV and ZIKV individually. A total of 29 ZIKV and 36 DENV single-infections were identified. Interestingly, 13 co-infection cases were observed, of which 8 were CHIKV/DENV, 3 were CHIKV/ZIKV, and 2 were DENV/ZIKV. There were six DENV genotypes . Additionally, ZIKV strains identified in this study either clustered with strains previously circulating in Thailand and Singapore, or with strains previously reported in China, French Polynesia, and the Americas. Our findings reveal the co-infection and genetic diversity patterns of mosquito-borne viruses circulating in Thailand. Dengue is the highly prevalent mosquito-borne viral disease transmitted primarily by Aedes spp. of mosquito, which also serve as vectors for CHIKV and ZIKV. DENV exhibits hyper-endemic transmission in rural and urban areas of Thailand, with an average of 50,000 cases per year2. The average dengue incidence rate in Thailand between 2018 and 2020 was 144 per 100,000 population, with 295 deaths3. Four antigenically distinct serotypes of DENV have been reported , and these serotypes exhibit 60–70% genome sequence similarity4. Each DENV serotype is further categorized into distinct genotypes depending on genetic divergence. Although all four DENV serotypes are generally found in dengue-endemic areas worldwide, genotypes within each virus serotype usually circulate in different geographic regions5. Both genotype replacement and co-circulation of multiple genotypes have been linked with virulence7.Important mosquito-borne viruses that cause human illness in Thailand include dengue virus (DENV), chikungunya virus (CHIKV), and Zika virus (ZIKV). Most people infected with DENV, ZIKV, or CHIKV share common symptoms, including high fever, rash, muscle pain, and joint painFlavivirus. ZIKV has probably circulated in Thailand at a low but sustained level since at least 20028. It is possible that ZIKV infection is commonly misdiagnosed as dengue fever or another illness due to the lack of a specific assay for ZIKV infection. However, a surge in the number of reported Zika fever cases in Thailand occurred in 2016–2017, with more than 1500 confirmed ZIKV infections9. It is plausible that this signaled the beginning of a large wave of ZIKV infections. After 2016–2017, Zika cases continued to be reported in Thailand, with 690, 142, and 144 confirmed cases in 2018, 2019, and 2020, respectively10. From 2016 to 2020, the Bureau of Epidemiology, Ministry of Public Health of Thailand reported 213 cases of pregnant women with confirmed ZIKV infection, including 11 women who had miscarriages, four of which were confirmed to be associated with ZIKV infection.ZIKV is closely related to DENV, as both are members of the genus Togaviridae, genus Alphavirus. CHIKV causes chikungunya fever (CHIKF). The first significant documented urban outbreak of chikungunya in Bangkok, Thailand, occurred in the 1960s11. From 2008 to 2009, a large CHIKV outbreak in Thailand affected mainly the southern region of Thailand, was driven by an albopictus-adaptive E1-A226V mutation in the East/Central/South African (ECSA) genotype, leading to approximately 54,000 infections. Ten years later, CHIKV infection re-emerged in Thailand, with > 27,000 cases reported by the end of 202012. Our previous study found that the re-emergence of CHIKV outbreaks in Thailand was sustained by circulation of strains harboring two mutations, E1-K211E and E2-V264A, in the E1-226A background13. These mutations were correlated with increased infectivity, dissemination, and transmission in Ae. aegypti14. Of a total of 1,806 samples from our previous cohort study, 1295 were confirmed as CHIKV-positive and 511 as CHIKV-negative.CHIKV is a mosquito-borne virus of the family 15. Surveillance of mosquito-borne viruses in endemic areas is necessary for enhance monitoring disease trends and identifying outbreaks. Therefore, this study aimed to investigate the prevalence of DENV, ZIKA and co-infection with DENV/ZIKV/CHIKV from the archived samples of a chikungunya cohort study of chikungunya suspected patients during the massive outbreak of CHIKV in Thailand from October 2018 to February 2020. We also characterized the genetic diversity of the detected DENV and ZIKV.In Thailand, CHIKV, DENV, and ZIKV co-circulate, and the symptoms of infection with these mosquito-borne viruses can be indistinguishable, resulting in underdiagnosis of specific infectionsp < 0.001), followed by fever (79.3%). Arthralgia and conjunctivitis of ZIKV mono-infection cases were found in 51.7 and 17.2%, respectively, were not significantly associated with the disease. Here, no neurological involvement was observed in any of the ZIKV-infected cases. Of those infected with DENV, 52.8% were male, while 47.2% were female. Gender was not statistically significant in both ZIKV and DENV mono-infection cases. The majority of DENV-confirmed cases were found in participants aged 21–30 (47.2%), followed by those aged 31–40 years (19.4%) and 41–50 years (13.9%), while the lowest prevalence was observed in patients aged 11–20 (2.6%). In this study, all DENV-infected cases represent fever. Arthralgia was reported in 52.8% of DENV-confirmed cases (p < 0.001). Rash was observed in 27.8% of DENV cases with no significant predictor of disease.ZIKV and DENV were initially screened using one-step real-time RT-PCR analysis of serum from the 511 CHIKV-negative patients. Of the 511 serum samples tested  based on differences in amino acids in three proteins, residue 17 in the prM protein, residue 188 in the NS1 protein, and residue 114 in the NS5 protein. Thus, the specific amino acid patterns in these three proteins of ZIKV Thai strains of 2019–2020 were then analyzed. Our ZIKV Thai strains of 2019–2020 were identified as SVM genotype, similar to previous Thai isolates and other Southeast Asian isolates. Some of the ZIKA Southeast Asian isolates and the Micronesian strain of 2007 were SAM genotype. By contrast, the ZIKV French Polynesian strains of 2013–2014 were NVM genotype, and all ZIKV isolates from the Americas contained the NS5-114 V sequence and were therefore classified as NVV genotype  from the distinct cluster shared the highest nucleotide similarity (99.64%) with a strain isolated from a Chinese tourist returning from Myanmar in 2019. The complete coding sequence of the Thai strain of 2020 (OM964568) also showed high nucleotide identity with sequences of ZIKV strains isolated in French Polynesia in 2013–2014 (99.07–99.16%) and strains reported previously in the Americas (98.77–99.07%), higher than the identity of previous Thai strains isolated between 2006 and 2017 (98.11–98.76%). According to a previous reportBased on analyses of 1806 patient serum samples obtained during our chikungunya cohort study from October 2018 to February 2020, 46 patients were confirmed by RT-PCR to have DENV infection  days and 3 (range 1–7) days, respectively  due to the inadequate volume of serum samples. We also assumed that DENV- and ZIKV-negative test results could be due to fever caused by other pathogens. A recent acute undifferentiated fever etiology study in Thailand reported that in addition to DENV, acute undifferentiated fever can also occur with murine typhus, leptospirosis, influenza, and bacteremia17.A total of 444 samples that were CHIKV negative by RT-PCR and IgM testing were also RT-PCR–negative for DENV and ZIKV. The timing of the test relative to disease stage is one factor that influences the accuracy of the diagnosis. The RT-PCR assay used here would have low sensitivity for serum samples collected late in the disease progression. Negative viral RNA detection results do not rule out a diagnosis of dengue or Zika fever because the viremic phase of DENV and ZIKV infection is short28. Mercado-Reyes et al. described seven fatalities related to DENV/CHIKV and CHIKV/ZIKV co-infection during epidemiologic monitoring of the ZIKV outbreak in Colombia between October 2015 and December 2016. Among the co-infection mortality cases, death was related to neurological manifestations, sepsis, and multiple organ failure in five cases, whereas the other two cases involved fetal death28. Nevertheless, of the co-infection cases in our study, fever was the most common clinical feature for all patients, and no severe symptoms were observed. Many previous studies have also reported that the clinical features of dual infections involving CHIKV/ZIKV29, CHIKV/DENV30, and DENV/ZIKV31 are not associated with worse outcomes and do not differ significantly in terms of clinical features compared with single infections with each virus. Moreover, one systematic review reported that only 9% of ZIKV–co-infected patients in cohort and cross-sectional investigations had complications32.A further significant finding of this study was the detection of 13 cases of co-infection, which comprised eight cases of CHIKV/DENV co-infection, three cases of CHIKV/ZIKV co-infection, and two cases of DENV/ZIKV co-infection. Some studies have reported that co-infection with mosquito-borne viruses can exacerbate disease severity33. Based on differences in the amino acid residues in three proteins−position 139 , 982 (188 in the NS1 protein), and 2634 (114 in NS5 protein)−the Asian lineage ZIKV isolates have been categorized into four major genotypes, SAM, SVM, NVM, and NVV16. Changes in a single amino acid residue at positions 139 and 982 are reportedly associated with more-severe disease and enhanced viral transmission35.In addition to diagnostic testing, investigations of viral genetic variations are essential for infection prevention and understanding how a virus spreads is necessary for further development of antiviral agents. In this study, phylogenetic analyses demonstrated that in Thailand, ZIKV belongs to the Asian genotype but forms two main clades. Most isolates clustered with previous Thai and Singapore strains isolated in 2016–2017. Isolates in the other clade were closely related to a ZIKV strain isolated in a Chinese traveler returning from Myanmar in 2019 as well as strains reported previously in French Polynesia and the Americas. Our findings are similar to those of a previous study that classified ZIKV from mosquitoes in 2016 in Thailand into two clades closely related to the Thai strain of 2013–2017 and the other clade related to viruses identified in the Americas36. Yuan et al.34 reported that the S139N substitution in prM enhances ZIKV replication in human and mouse neural progenitor cells, resulting in more-severe microcephaly in infected mouse fetuses. The A982V substitution in the NS1 protein increases ZIKV NS1 secretion into the host circulation and substantially enhances transmission of ZIKV in mosquito vectors35. Additionally, the NS1 A982V mutation enhances ZIKV replication via inhibition of interferon-β induction37. A change of methionine or threonine to valine at position 2634 (M//T2634V) from the start codon of the genome or residue 114 in the NS5 protein is a unique molecular hallmark of all American strains isolated in 2015–201638.ZIKV exhibits a serine to asparagine substitution at position 139 (S139N) in prM, which arose during the ZIKV outbreak that occurred in early 2013 in French Polynesia before spreading throughout the Americas36. It could be hypothesized that the M2634V substitution in the American strains is responsible for the increased outbreak potential of the mutant virus in the Americas. However, recent studies have shown that the insignificant M2634V mutation in the NS5 protein enhances viral replication and transmission potential, indicating that the M2634V substitution is not responsible for enhancing the ZIKV epidemic potential40. Here, we found that the ZIKV Thai strains isolated in 2019–2020 were SVM, similar to most previously isolated Thai and other Southeast Asian strains. Although the SVM strains do not carry the S139N mutation, which is associated with increased neurovirulence of ZIKV34, it was reported that infection with SVM ZIKV variants can lead to congenital Zika syndrome and microcephaly. However, one crucial factor in the development of microcephaly in a fetus or infant is the timing of ZIKV infection during pregnancy. A high risk of microcephaly with ZIKV infection is observed in the first trimester41.In contrast, strains from Southeast Asia, the Pacific, and Africa harbor 2634M, and a ZIKV Malaysian strain isolated in 1966 harbored 2634T42. All DENV-1 isolates detected in this study belonged to genotype I, which was first documented in Thailand in 198143. Since that time, DENV-1 genotype I has been routinely isolated in Thailand and several other countries in Southeast Asia45. Interestingly, our DENV-1 phylogenetic tree showed that previous and present genotype I Thai strains did not cluster in the same clade. Most of the Thai strains in each clade were closely related to genotype I strains from neighboring countries, demonstrating intra-genotypic variation of DENV-1 genotype I strains in Thailand and a genetic link between DENV-1 circulating in Thailand and other countries in Southeast Asia. We also observed geographic expansion of DENV-1 genotype I strains from Southeast Asia to East Asia as a result of the close relationship between Thai and Cambodian with DENV-1 isolated from China. A previous report also showed that an epidemic involving DENV-1 in China was related to infected Chinese tourists returning from Thailand46.Among DENV-positive samples, all four serotypes were detected in this study, indicating that all four DENV serotypes co-circulated in Thailand in 2019–2020. The most common serotype was DENV-2, consistent with a 2020 report from the Bureau of Vector-Borne Diseases, Department of Disease Control, Ministry of Public Health, Thailand. In this study, six different DENV genotypes were identified among the four serotypes. DENV-1 comprises genotypes I, II, III, IV, and V47. The sylvatic genotype circulates in West Africa and Southeast Asia and frequently exhibits enzootic cycles between forest mosquitoes and nonhuman primates. The American genotype is present in South and Central America. Over the past three decades, the Asian/American genotype circulating in the Americas has caused several epidemics in South and Central America48. This genotype was previously the dominant circulating genotype in Southeast Asia, but Asian I genotype viruses began to replace the Asian/American genotype in 199249. The Asian I genotype of DENV-2 is frequently detected in many Asian countries51. The Asian II genotype includes strains from Asia, among which the DENV-2 Cosmopolitan genotype is the most prevalent globally52. A phylogenetic analysis of DENV-2 isolates based on envelope region sequence indicated that both the Asian I and Cosmopolitan genotypes co-circulated in Thailand during 2019–2020, especially in Bangkok. Previous studies also found that both genotypes of DENV-2 co-circulated in Southern Thailand in 2015–201645.Six distinct genotypes of DENV-2 have been identified53. DENV-3 genotype II was the predominant genotype in Thailand in the 1970s54. Displacement of genotype II by genotype III in Thailand was reported in 200855. DENV-3 genotype I was first documented in 198856. In 2015, DENV-3 genotype I re-emerged and co-circulated with DENV-3 genotype III in southern Thailand, but genotype III was detected more frequently than genotype I45. In contrast, other studies have reported that only genotype III circulated in Thailand in 2016–201757. The present study found that DENV-3 genotypes I and III co-circulated in Bangkok in 2019–2020, with genotype I detected at higher frequency than genotype III. This evidence suggests there has been a genotypic shift in DENV-3 in Thailand. A study in Myanmar, a neighboring country of Thailand, also reported co-circulation of the emerging genotype I in that country from 2017 to 201958. Our phylogenetic analysis showed that DENV-3 genotype I is closely related to DENV-3 strains from Myanmar, indicating the possibility of the virus spreading across countries. In addition, our DENV-3 genotype III formed two phylogenetic clusters, one closely related to strains from Myanmar and the other related to Indian strains, indicating the spread of the virus between countries in Southeast Asia and South Asia.Five DENV-3 genotypes (I-V) have been identified to dateDENV-4 was rarely isolated in this study. Phylogenetic analysis revealed that the DENV-4 Thai strains isolated in 2019–2020 were predominantly of genotype I and closely related to the DENV-4 isolate from Myanmar and a Chinese traveler who visited Myanmar suggesting the spread of the virus to a nearby country and wider geographic expansion of the virus from Southeast Asia to East Asia.In conclusion, we found that cases of dengue fever and Zika infection were underestimated and underreported during the large CHIKV outbreak that occurred in Thailand between October 2018 and February 2020. A possible reason for this underestimation and underreporting is that infections involving CHIKV, DENV, and ZIKV can cause similar but widely varying clinical presentations. As such, accurate laboratory diagnosis is imperative. We thus highlight the co-infection and co-circulation of these three mosquito-borne viruses in Thailand. ZIKV circulation in Thailand during 2019–2020 was the Asian genotype divided into two clades: one group related to previous circulating Thai and Singaporean strains, and the other closely related to viruses identified in China, French Polynesia, and the Americas. Four DENV serotypes were detected, including six genotypes consisting of 13 DENV-1 genotype I, 10 DENV-2 Asian I, 10 DENV-2 Cosmopolitan, 6 DENV-3 genotype I, 2 DENV3 genotype III, and 5 DENV-4 genotype I. Notably, most of the genotypes were also classified into multiple clades. Most confirmed cases occurred in Bangkok, the capital and most populous city in Thailand. In addition, molecular characterization of the viral genomes revealed the degree of genetic variation between ZIKV and the four DENV serotypes. Bangkok undoubtedly serves as the epicenter for mosquito-borne virus diversity in Thailand. Continuous monitoring of viral genetics, in conjunction with development of a surveillance database, may enhance our understanding of viral genotype changes and their association with epidemiology, thereby helping prevent or control outbreaks involving these viruses.The study was carried out in compliance with the guidelines of the Institutional Review Board of the Ethics Committee of the Chulalongkorn University Faculty of Medicine, which evaluated and approved the study (IRB no. 710/64). The Institutional Review Board of the Ethics Committee for Human Research waived the need for informed consent from participants because all clinical specimens were de-identified. All experiments were performed following relevant guidelines and regulations.13. The present study examined 1806 serum samples from the previous cohort study collected from patients with suspected CHIKV infection from 13 provinces of Thailand between October 2018 and February 2020. A suspected case of chikungunya involves a patient with acute onset of fever, often reaching more than 38.5 °C, with or without arthralgia and rash, especially in a person who has resided or visited a known risk area of CHIKV transmission. The age of subjects varied from 3 to 97 years. In patients with suspected CHIKF, serum samples were collected and examined for the presence of CHIKV, as described previously13. CHIKV infection was confirmed in 1295 (71.7%) of 1806 samples by positive real-time reverse transcriptase–polymerase chain reaction (RT-PCR) results and/or CHIKV IgM antibody, and 511 (28.3%) samples were negative for CHIKV. All samples were re-evaluated to detect ZIKV and DENV by using RT-PCR.This work retrospectively investigated serum samples used in a previous study60. Amplifications were conducted on an ABI viiA7 Real-Time PCR instrument (Applied Biosystems).Total RNA was extracted from 200 μL of each sample using a magLEAD® kit. To screen for ZIKV and DENV, qualitative one-step real-time RT-PCR was performed with specific primer sets and fluorophore-labeled hybridization probes using previously described PCR conditions61, with some modifications. In brief, RNA was used as the template in a total reaction volume of 20 µL of SensiFAST reagent  with dengue consensus forward primer (D1: 5’-TCAATATGCTGAAACGCGCGAGAAACCG-3’) and four serotype-specific reverse primers for DENV-1 (Ts1: 5’-CGTCTCAGTGATCCGGGGA-3’), DENV-2 (Ts2: 5’-CGCCACAAGGGCCATGAACAG-3’), DENV-3 (Ts3: 5’-TAACATCATCATGAGACAGAGC-3’), and DENV-4 (Ts4: 5’-CTCTGTTGTCTTAAACAAGAGA-3’). The reaction mixture was subjected to the following cycling conditions: cDNA synthesis step at 45 °C for 20 min, initial denaturation at 94 °C for 2 min, followed by 40 replication cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 45 s, extension at 72 °C for 45 s, and final extension at 72 °C for 5 min. The amplification products were analyzed by 2% agarose gel electrophoresis. Samples positive for DENV-1, DENV-2, DENV-3, or DENV-4 were determined based on detection of a PCR product of 482, 119, 290, or 392 bp, respectively.All DENV-positive samples were subjected to serotype analysis using conventional one-step DENV serotype-specific multiplex RT-PCR according to a previous report62. For DENV characterization, RNA extracted from serum of confirmed dengue cases with serotype identification was used as the template to amplify the dengue C-prM and E genes. One-step RT-PCR was performed with primers previously designed by Diaz-Badillo et al.61 and Cruz et al.63 to amplify the C-prM and E genes, respectively. Amplicons were purified using an Expin™ Combo GP kit  according to the manufacturer’s instructions. The purified products were then subjected to Sanger sequencing . The resulting nucleotide sequences were analyzed using Chromas Lite version 2.0 and the NCBI Basic Local Alignment Search Tool (BLAST) (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Edited nucleotide sequences were assembled manually using BioEdit Sequence Alignment Editor Software, version 7.0.5.3. All nucleotide sequences were aligned using MUSCLE (multiple sequence comparison by log expectation), implemented in the MEGA 11 program. A maximum-likelihood tree was constructed using the IQ-TREE tool (http://iqtree.cibiv.univie.ac.at/) with optimal model selection and 1000 ultrafast bootstrap replicates. Finally, the constructed phylogenetic tree was visualized and edited using FigTree v.1.4.4 software. All viral gene sequences were deposited in the NCBI GenBank. The ZIKV structural region sequences (C-prM-E) were deposited in GenBank under accession numbers OM936182-OM936195, complete ZIKV coding sequence (accession numbers: OM964565-OM964568), DENV-1 partial E region (accession numbers: ON780003-ON780014), DENV-2 partial E region (accession numbers: ON782110-ON782129), DENV-3 partial prM region (accession numbers: ON799425-ON799432), DENV-4 partial E region (accession numbers: ON787752-ON787756).For ZIKV, amplification of the structural protein gene (C-prM-E) and coding-complete genome sequence were performed using a Superscript III one-step RT-PCR system  with previously reported primers and PCR conditions p value of less than 0.05 was considered as statistically significant.The qualitative data were shown as n (%) and the mean with standard deviation for the day of disease onset detection and age of the patients. The chi-square test and logistic regression analysis were performed to compare demographic variables and the presence of symptoms among different groups of infections. Statistical analysis of significance was analyzed by using SPSS software, version 22. A Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.


High-density lipoprotein (HDL) is a heterogeneous group of subpopulations differing in protein/lipid composition and in their anti-atherogenic function. There is a lack of assays that can target the functionality of HDL particles related to atherosclerosis. The objective of this study was to construct two-site apolipoprotein A-I (apoA-I) assays and to evaluate their clinical performance in patients with suspected obstructive coronary artery disease (CAD).109−121 and apoA-I110−525 were measured in 197 patients undergoing coronary computed tomography angiography (CTA) and myocardial positron emission tomography perfusion imaging due to suspected obstructive CAD. Among patients not using lipid-lowering medication , the level of apoA-I110−525 was higher in the presence than in the absence of coronary atherosclerosis , whereas there was no difference in apoA-I109−121, HDL cholesterol, and apoA-I determined using a polyclonal apoA-I antibody. The levels of apoA-I109−121 and apoA-I110−525 were similar in the presence or absence of obstructive CAD. Among patients not using LLM, apoA-I110−525 adjusted for age and sex identified individuals with coronary atherosclerosis with a similar accuracy to traditional risk factors [area under the curve [AUC] (95% CI): 0.75(0.66–0.84) 0.71 (0.62–0.81)]. However, a combination of apoA-I110−525 with risk factors did not improve the accuracy [AUC (95% CI): 0.73 (0.64–0.82)].Direct two-site apoA-I assays (named 109–121 and 110–525) were developed to identify the presence of apoA-I in the HDL of patients with CAD using apoA-I antibodies as a single-chain variable fragment fused with alkaline phosphatase. ApoA-IDirect two-site apoA-I assays recognizing heterogeneity in reactivity with apoA-I could provide a potential approach to identify individuals at a risk of coronary atherosclerosis. However, their clinical value remains to be studied in larger cohorts. Atherosclerotic cardiovascular diseases (ASCVDs) remain as major causes of morbidity and mortality throughout the world . Plasma via its protein and/or lipid components  that could specifically target HDL functionality and could be used to improve the risk estimation of ASCVD. The production of mAbs through conventional animal immunization against a complex molecule like HDL is challenging. The phage display-based universal recombinant antibody libraries provide a platform where antibodies can be produced against almost any target . RecentlWe have previously developed phage-based two-site apoA-I assays (assay 109–121 and assay 110–525) by using phage-displayed single-chain variable fragment (scFv) antibodies  isolatedN = 21), missing information on the use of lipid-lowering medication , a failed or non-diagnostic coronary CTA (N = 11), or indications of coronary CTA other than an evaluation of a suspected CAD (N = 7). Hence, 197 samples remained for clinical evaluation of the direct two-site apoA-I assays.We prospectively recruited 252 symptomatic patients referred to Turku University Hospital for coronary CTA due to suspected obstructive CAD from March 2016 to January 2019 . Blood sThe clinical characteristics of the patients and findings of invasive coronary angiography (ICA) were collected from electronic medical records. The Framingham Risk Score (FRS) was utilized in the calculation of a 10-year coronary heart disease (CHD) risk . As predN = 200). Some of these samples were used as assay controls during the optimization and validation of the direct two-site apoA-I assays.Serum samples of randomly selected and routinely analyzed non-cardiac patients from Helsinki University Hospital (Finland) were also collected , as previously described , 25. Bef15O-water tracer during adenosine stress to assess the hemodynamic significance of the stenosis. In our cohort, 47 patients underwent a PET perfusion study, whereas eight patients with an obstructive CAD on the CTA were directly referred to an ICA due either to a contra-indication to the PET perfusion study (N = 5) or to logistic reasons (N = 3).As previously described , in the 15O-water  was injected as an intravenous bolus (injected activity of 500–600 MBq) over 15 s, and a dynamic PET acquisition was performed .Scans were performed after an overnight fast. Patients were instructed to abstain from alcohol and caffeine for 24 h before the PET myocardial perfusion study. The adenosine infusion was started 2 min before the stress PET scan and continued at a rate of 140 μg/kg/min until the scan was complete. The coronary CTA images were analyzed according to the 17-segment vessel system using the GE ADW 4.4 Workstation software . The presence of coronary atherosclerosis and the diameter of the stenosis were evaluated in all segments. An Agatston coronary artery calcium score was measured in the non-enhanced scan.The PET data were analyzed quantitatively using the Carimas software  , 26. AbsIn this study, obstructive CAD was defined as the presence of atherosclerosis on coronary CTA accompanied by an abnormally stressed MBF or, alternatively, the presence of > 70% stenosis on the ICA. The rest of the patients were classified as having a non-obstructive CAD or normal coronary arteries (no atherosclerosis) based on the CTA findings.2 + HDL3 subclasses) from the serum of a healthy individual was isolated, as described earlier (+3) chelate of tetra-tert-butyl 2,2′,2″,2‴--tetraacetate (referred to as Eu+3-WN) was synthesized according to Wang et al.  at 4°C, overnight with shaking using a modified protocol of Eriksson et al. , triglycerides (TG), TC, PL transfer protein (PLTP) activity, paraoxonase activity (PON-I), and serum-free glycerol. TG and TC were measured using enzymatic methods . HDL-C was analyzed using a phosphotungstate-MgClfugation . ApoA-I fugation . Brieflyfugation . LDL-C wfugation . PON-1 afugation . The serTwo distinct pairs of recombinant scFv apoA-I antibodies (sc 109–121 and sc 110–525), which were previously tested with an optimized phage-based two-site apoA-I immunoassay and had shown diagnostic/predictive value for MI and mortality , were us+3-WN labeled detection antibodies (scFv-AP) sc 121 and sc 525, respectively. Assay 109–121 was done with an HDL-assay buffer supplemented with 4% BSA and assay 110–525 was performed with an HDL-assay buffer containing 2.3% BSA on streptavidin plates. The total HDL , i.e., HDL2 + HDL3 subpopulations isolated from the serum of a healthy individual was used as an assay calibrator or standard.The final optimized assays used the scFv-AP apoA-I antibody pairs 109–121 and 110–525. A schematic representation of the principle of these direct two-site apoA-I assays is shown in +3-WN labeled detection antibodies sc 121 (100 ng/50 μl/well) and sc 525 (200 ng/50 μl/well) were added to the respective assays and incubated for 1 h at RT with shaking. Finally, the wells were washed four times. EFI (200 μl/well) was added to the well, incubated for 10 min with shaking, and the TRF of the europium was measured with a Victor plate reader .Biotinylated-captured antibodies sc 109 and sc 110 (100 ng/50 μl/well) were immobilized on separate streptavidin-coated microtiter plates and incubated for 1 h at room temperature (RT) with shaking followed by two washes. For both assays, a calibrator and sample were added in four replicates and incubated for 1 h at RT with shaking. After two washes, the EuN = 15 for the blanks and N = 12 per level for the standard in assays 109–121, and N = 20 for the blanks and N = 16 per level for the standard in assay 110–525. The analytical sensitivity was calculated by adding five times the standard deviation (SD) to the average count for the blank sample (blank + 5*SD) and performing a linear fitting between the concentration of the calibrator and the corresponding average counts. Within 3 days, the inter-assay variation of the assays was tested with eight routinely analyzed serum samples , using four replicates (N) per sample, i.e., N = 12 per sample. The linearity of the assay was assessed by diluting the four serum samples  from 62.5- to 1,000-fold, using three replicates per sample (N = 3).The analytical sensitivity of the direct two-site apoA-I assays was evaluated by running eleven levels of the standard  and the blanks (HDL = 0 ng/ml) through several replicates, i.e., N = 197). Comparisons were done between the following groups . The normality distribution was checked with a Shapiro–Wilk test and a Q-Q plot. Categorical variables are presented as numbers (percentage) and analyzed by a chi-squared test or a Fisher's exact test, as appropriate. Continuous variables are presented as a median . The concentration of the apoA-I obtained from the direct two-site apoA-I assays and the concentration of biochemical parameters were used after a natural log transformation was performed in order to enable parametric statistical testing.The statistical analysis was done using Origin 2015 , JMP Pro 13 , and IBM110−525 or apoA-I109−121 levels with age, with the Agatston coronary calcium score and with conventional apoA-I ELISA . Biochemical parameters and the FRS for the CHD were compared between the two groups (no atherosclerosis vs. atherosclerosis and obstructive CAD vs. non-obstructive atherosclerosis) by using an independent t-test. For a comparison between the three groups , a one-way ANOVA was used followed, if required, by a multiple pair comparison using a Tukey's test.A suitable Pearson's or Spearman's correlation was used to test the correlation of apoA-I110−525 or apoA-I109−121 levels combined with age and gender (Model 1) or age, gender, smoking, diabetes, hypertension, LDL-C, and HDL-C (Model 2) as the independent variables. The apoA-I concentrations obtained from the two-site apoA-I assays were used as a categorical variable (below and above the median). An analysis of the area under the receiving-operating characteristic (ROC) curve was performed to test the diagnostic clinical value of the apoA-I110−525 or apoA-I109−121 levels and the FRS CHD. The ROC curves were compared using the DeLong's method. The results were analyzed for the whole cohort as well as separately in patients using and not using LLM. All the test results were considered statistically significant for P-value of < 0.05.A logistic regression analysis was performed to estimate the odds ratios (ORs) and 95% confidence intervals (CIs), with atherosclerosis and obstructive CAD as the dependent variables, and the apoA-Ir2 = 0.98) and 6.8–2,564 ng/ml (r2 = 0.99). The inter-assay variation within 3 days with assay 109–121 was between 2–9% and 4–16% in the standards and samples; the inter-assay variation with apoA-I assay 110-525 in the standards and samples was 3–22% and 7–26%, respectively (r2 = 0.99) and 250- to 1,000-fold (r2 = 0.99) was suitable for assays 109–121 and 110–525, respectively.The standard curves of the direct two-site apoA-I assays where the X-axis represents the HDL protein concentration  and the Y-axis represents the average counts  of the standard are shown in ectively . The intThe median age of the 197 patients was 63 years and 43% were males. The most common symptom was atypical chest pain (52%). The baseline characteristics of the patients are displayed in P = 0.007) and PON-I  were higher in patients without any coronary atherosclerosis than in those with atherosclerosis (P = 0.01) and LDL-C (P = 0.01) were higher in patients without obstructive CAD than in those with obstructive CAD (P = 0.03) and PLTP (P = 0.04) were significantly lower in patients with obstructive CAD than in those without obstructive CAD (P < 0.0001) in patients with coronary atherosclerosis than in those with normal coronary arteries  did not show any significant correlation (Pearson's r) with a conventional ELISA-based apoA-I assay  showed a weak positive correlation  with each other , since apoA-I110−525 tended to be lower in the presence of atherosclerosis with a borderline significance (P = 0.05) among patients taking LLM.Compared to patients without coronary atherosclerosis, the level of apoA-I109−121 was significantly higher in patients without any coronary atherosclerosis than in those with coronary atherosclerosis among LLM users (P = 0.03) but not in patients not taking LLM (P = 0.88) or in the whole cohort  with the extent of coronary atherosclerosis measured by the coronary calcium score.Neither of the two direct apoA-I assays showed a correlation  and separately in LLM users (P = 0.14) and non-LLM users  and separately in LLM users (P = 0.22) and non-LLM users  (110−525 between patients with non-obstructive and obstructive CAD (P = 0.16) or obstructive CAD and without atherosclerosis (P = 0.99). ApoA-I109−121  could not discriminate between any of these groups (We further compared the concentration of apoA-It cohort  and sepat cohort . Among i = 0.01) . Howevere groups .109−121 level was higher in females than in males  as well as age, sex, and risk factors for CAD  in patients not using LLM : 0.72 (0.65–0.80) vs. 0.64 (0.57–0.72)] in the whole population, [0.64 (0.50–0.78) vs. 0.55 (0.40–0.69)] in LLM users, and [0.75 (0.66–0.84) vs. 0.71 (0.62–0.81)] in patients not using LLM  : 0.65 (0.57–0.72) vs. 0.64 (0.57–0.72)] in the whole population, [0.60 (0.46–0.75) vs. 0.55 (0.41–0.69)] in LLM users, and [0.73 (0.64–0.82) vs. 0.71 (0.62–0.81)] in patients not using LLM  (ApoA-I > 0.05) . ApoA-I1 > 0.05) .An ROC curve was not drawn for the prediction of atherosclerosis using the direct two-site apoA-I assay 109–121 since the data from this assay were not significantly associated with atherosclerosis in the logistic regression analysis .2 and HDL3 subpopulation from normolipidemic subjects) was used as the calibrator since the apoA-I reactive epitopes of these anti-apoA-I antibodies were unknown. The optimized assays were very sensitive and a serum dilution of up to 1,000-fold was found to be sufficient for these assays. We evaluated whether differences detected by the new recombinant apoA-I antibodies and the respective direct two-site apoA-I assays are related to coronary atherosclerosis and its severity in patients with suspected obstructive CAD.This study presents the development and clinical evaluation of time-resolved fluorescence (TRF) based on direct two-site apoA-I immunoassays (assays 109–121 and 110–525); the assays were established using recombinant apoA-I antibodies previously generated against the intact isolated HDL particles from plasma of patients with CAD . The apo109−121 ) and assay 110–525 (referred to as apoA-I110−525) was significantly higher in females compared to males. We found a positive correlation between direct two-site apoA-I assays and age specifically in patients not using LLM. The direct two-site apoA-I assays 110–525 did not show any significant correlation with a conventional ELISA-based apoA-I test; however, the assays 109–121  showed a weak positive correlation. It is worth noting that the ELISA-based apoA-I assay was performed using polyclonal antibodies against purified apoA-I  is widely used for the prevention of cardiovascular disease by reducing LDL-C and TG  and its d apoA-I . Howevervia the function of PON-1 is compromised in these subjects. The PON-1 function is physiologically important to attenuate the LDL oxidation generating minimally modified oxLDL which is related to atherogenesis , only the total PL serum and PON-I activity were significantly lower in patients with coronary atherosclerosis, which is in accordance with previous studies , 38. Theogenesis . Notablyogenesis .110−525 was associated with coronary atherosclerosis in patients not using LLM. This association was present in multivariable models including age, gender, and traditional CAD risk factors. We also tested whether apoA-I110−525 could improve the identification of patients with coronary atherosclerosis as compared to traditional risk factors. FRS is a well-established method to evaluate the 10-year risk of having CHD  in individuals not using LLM than in those taking LLM. Whether this approach could be used to estimate the likelihood of coronary atherosclerosis in LLM naive individuals remains to be tested in a larger cohort. In contrast to apoA-I110−525, apoA-I109−121 was lower in the presence of atherosclerosis among LLM users, indicating that these antibodies recognize different epitopes. The difference in the apoA-I109−121 levels was not significant when the risk factors were included.Previous studies have demonstrated that a low level of serum apoA-I is associated with CAD and MI , 42). O. O42). Oving CHD . ApoA-I1The amount of coronary artery calcium reflects the extent of the coronary atherosclerosis and allows a risk prediction for the general population . The twoThere are some limitations to this study, which must be considered. Due to the small number of patients, the findings may be viewed as exploratory, but they demonstrate the proof-of-concept that recombinant apoA-I antibodies derived from the phage display library against CAD HDL particles may be used to improve the estimation of the risk of coronary atherosclerosis. However, the findings need to be confirmed in a larger cohort. With regard to the antibodies used in the direct two-site assays, they have already been characterized for their various properties as a part of the study where they were discovered and further investigated , 19. SDSIn summary, TRF-based direct two-site apoA-I immunoassays were developed using novel recombinant apoA-I antibody pairs (sc 109–121 and sc 110–525). The assays were clinically evaluated and compared with other traditional risk predictors  with a well-characterized clinical cohort of patients with suspected obstructive CAD. In patients not using LLM, a high level of apoA-I identified with the antibody pair 110–525 was associated with the presence of coronary atherosclerosis; however, HDL-C and apoA-I levels measured using a polyclonal anti-apoA-I-based ELISA test were not. The direct two-site apoA-I assay 110–525 showed a similar prediction of atherosclerosis to FRS. In conclusion, assays targeting heterogeneity in HDL's main apolipoprotein, apoA-I, could provide a potential approach to improving the identification of patients with a risk of developing coronary atherosclerosis.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Local Ethics Committee of the Hospital District of Southwest Finland. The patients/participants provided their written informed consent to participate in this study.KP, JL, UL, MJ, JK, and AS contributed to the study concept and design. PN, TH, KV, and JM performed the laboratory work. PN and AS contributed to data analysis. AS, ET, WN, TM, and JK contributed to the collection of patients' samples or medical information or image acquisition and interpretation. PN, AS, and JM wrote the first draft of the manuscript. All authors contributed to the article and approved the submitted version.This research was supported by the Doctoral Program of Clinical Investigation (CLIDP), University of Turku Graduate School (to PN), the Academy of Finland , and the Finnish Foundation for Cardiovascular Research (to MJ and AS).Author AS declares fees for lectures or consultancy from Abbott, Amgen, Astra Zeneca (AS and JK), Boehringer Ingelheim, Bayer and Pfizer, Novartis (JK), GE Healthcare (JK). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.